Diabetic retinopathy remains a leading cause of blindness in the United States with no FDA approved medical therapy. The goal of this proposal is to understand the molecular mechanisms that lead to increased vascular permeability and macular edema and to elucidate the relationship of vascular permeability to angiogenesis so that novel therapies to treat or prevent diabetic retinopathy may be developed. Vascular dysfunction in diabetic retinopathy may result from both the direct effect of hyperglycemia, advanced glycation end products, and hyperlipidemia on vascular endothelial cells and the indirect effect of these metabolites through induction of growth factors such as vascular endothelial growth factor (VEGF) and inflammatory cytokines such as tumor necrosis factor (TNF). In the current proposal, it is hypothesized that VEGF and TNF alter the tight junction complex leading to increased endothelial permeability. Research over the previous funding period has demonstrated that VEGF induces phosphorylation of the tight junction protein occludin in a protein kinase C dependent manner that is associated with vascular permeability. Further, VEGF induces redistribution of occludin and other tight junction proteins from the plasma membrane to the cell cytoplasm and over time, leads to the degradation of occludin. Analysis of occludin phosphorylation sites by mass spectrometry has identified VEGF responsive phospho-sites. In this proposal, data is presented demonstrating that mutation of occludin to prevent phosphorylation, blocks VEGF-induced permeability and occludin endocytosis. Furthermore, preliminary data demonstrate that TNF alters the tight junction complex by reducing the tight junction proteins claudin 5 and zonula occludens 1 but does not decrease occludin content suggesting at least partially divergent mechanisms. Occludin content is closely associated with proliferation of cells that possess tight junctions. Silencing occludin expression in retinal pigment epithelium cells induces a two-fold increase in cell proliferation. Therefore, we will examine the mechanisms by which VEGF and TNF alter the tight junction complex to induce endothelial permeability and the relationship of VEGF-induced occludin phosphorylation and degradation to angiogenesis. While VEGF and TNF diverge in control of occludin, preliminary data demonstrates both factors utilize the atypical PKC pathway to alter the junctional complex and induce endothelial permeability. Therapies targeting atypical PKC pathway alone or in conjunction with PKC inhibitors may provide an effective means to control vascular permeability in diabetic retinopathy and other retinal diseases involving VEGF and inflammatory cytokines.